目的 探讨缺血后处理激活大鼠心肌缺血再灌注时NF-E2相关因子2(Nrf2)-抗氧化反应元件(ARE)信号通路的机制与活性氧(ROS)的关系.方法 健康雄性SD大鼠,体重250～300g,16～20周龄,采用Langendorff灌注装置建立大鼠离体心脏灌注模型.取模型制备成功的心脏32个,采用随机数字表法分为4组(n=8):对照组(C组)、缺血再灌注组(I/R组)、缺血后处理组(IPO组)和ROS清除剂N-(2-巯基丙酰)-甘氨酸+缺血后处理组(M+IPO组).平衡灌注20 min后,C组继续灌注100 min;I/R组灌注4℃St.Thomas停跳液停跳,并在32℃下缺血40 min,再灌注60 min;IPO组于再灌注即刻行缺血后处理,再灌注10 s,缺血10 s,共6个循环,然后恢复灌注58 min;M+IPO组于再灌注即刻灌注含N-(2-巯基丙酰)-甘氨酸2 mmol/L的K-H液3 min,然后行缺血后处理2min,再灌注55 min.分别于平衡灌注末及再灌注末记录HR、左心室发展压(LVDP)、左心室舒张末压(LVEDP)和左心室压力最大上升速度(+dp/dtmax).分别于再灌注5 min和再灌注末时,取左心室心肌组织,采用ELISA法测定ROS含量.于再灌注末,取左心室心肌组织,观察心肌细胞超微结构,并进行线粒体损伤评分,分别采用Western blot法和RT-PCR法检测心肌组织Nrf2、血红素加氧酶-1(HO-1)、醌氧化还原酶1(NQO1)、超氧化物歧化酶1(SOD1)及其mRNA的表达水平.结果 与C组比较,再灌注末I/R组和M+IPO组HR、+dp/dtmax和LVDP降低,LVEDP升高,IPO组HR、LVDP降低,LVEDP升高(P＜0.05),HR和+dp/dtmax差异无统计学意义(P＞0.05),I/R组、IPO组和M+IPO组线粒体损伤评分升高,再灌注末I/R组、IPO组和M+IPO组ROS含量升高,心肌组织Nrf2、HO-1、NQO1和SOD1及其mRNA表达下调(P＜0.05).与I/R组比较,IPO组和M+IPO组再灌注末HR、+dp/dtmax和LVDP升高,LVEDP和心肌组织ROS含量降低,心肌组织Nrf2、HO-1、NQO1和SOD1及其mRNA表达上调,IPO组线粒体损伤评分降低(P＜0.05),M+IPO组线粒体损伤评分差异无统计学意义(P＞0.05).与IPO组比较,M+IPO组再灌注末HR、+dp/dtmax和LVDP降低,LVEDP和心肌组织ROS含量升高,线粒体损伤评分升高,心肌组织Nrf2、HO-1、NQO1和SOD1及其mRNA表达下调(P＜0.05).结论 缺血后处理可调节ROS的水平,激活Nrf2-ARE信号通路,减轻大鼠心肌缺血再灌注损伤.
目的 探討缺血後處理激活大鼠心肌缺血再灌註時NF-E2相關因子2(Nrf2)-抗氧化反應元件(ARE)信號通路的機製與活性氧(ROS)的關繫.方法 健康雄性SD大鼠,體重250～300g,16～20週齡,採用Langendorff灌註裝置建立大鼠離體心髒灌註模型.取模型製備成功的心髒32箇,採用隨機數字錶法分為4組(n=8):對照組(C組)、缺血再灌註組(I/R組)、缺血後處理組(IPO組)和ROS清除劑N-(2-巰基丙酰)-甘氨痠+缺血後處理組(M+IPO組).平衡灌註20 min後,C組繼續灌註100 min;I/R組灌註4℃St.Thomas停跳液停跳,併在32℃下缺血40 min,再灌註60 min;IPO組于再灌註即刻行缺血後處理,再灌註10 s,缺血10 s,共6箇循環,然後恢複灌註58 min;M+IPO組于再灌註即刻灌註含N-(2-巰基丙酰)-甘氨痠2 mmol/L的K-H液3 min,然後行缺血後處理2min,再灌註55 min.分彆于平衡灌註末及再灌註末記錄HR、左心室髮展壓(LVDP)、左心室舒張末壓(LVEDP)和左心室壓力最大上升速度(+dp/dtmax).分彆于再灌註5 min和再灌註末時,取左心室心肌組織,採用ELISA法測定ROS含量.于再灌註末,取左心室心肌組織,觀察心肌細胞超微結構,併進行線粒體損傷評分,分彆採用Western blot法和RT-PCR法檢測心肌組織Nrf2、血紅素加氧酶-1(HO-1)、醌氧化還原酶1(NQO1)、超氧化物歧化酶1(SOD1)及其mRNA的錶達水平.結果 與C組比較,再灌註末I/R組和M+IPO組HR、+dp/dtmax和LVDP降低,LVEDP升高,IPO組HR、LVDP降低,LVEDP升高(P＜0.05),HR和+dp/dtmax差異無統計學意義(P＞0.05),I/R組、IPO組和M+IPO組線粒體損傷評分升高,再灌註末I/R組、IPO組和M+IPO組ROS含量升高,心肌組織Nrf2、HO-1、NQO1和SOD1及其mRNA錶達下調(P＜0.05).與I/R組比較,IPO組和M+IPO組再灌註末HR、+dp/dtmax和LVDP升高,LVEDP和心肌組織ROS含量降低,心肌組織Nrf2、HO-1、NQO1和SOD1及其mRNA錶達上調,IPO組線粒體損傷評分降低(P＜0.05),M+IPO組線粒體損傷評分差異無統計學意義(P＞0.05).與IPO組比較,M+IPO組再灌註末HR、+dp/dtmax和LVDP降低,LVEDP和心肌組織ROS含量升高,線粒體損傷評分升高,心肌組織Nrf2、HO-1、NQO1和SOD1及其mRNA錶達下調(P＜0.05).結論 缺血後處理可調節ROS的水平,激活Nrf2-ARE信號通路,減輕大鼠心肌缺血再灌註損傷.
목적 탐토결혈후처리격활대서심기결혈재관주시NF-E2상관인자2(Nrf2)-항양화반응원건(ARE)신호통로적궤제여활성양(ROS)적관계.방법 건강웅성SD대서,체중250～300g,16～20주령,채용Langendorff관주장치건립대서리체심장관주모형.취모형제비성공적심장32개,채용수궤수자표법분위4조(n=8):대조조(C조)、결혈재관주조(I/R조)、결혈후처리조(IPO조)화ROS청제제N-(2-구기병선)-감안산+결혈후처리조(M+IPO조).평형관주20 min후,C조계속관주100 min;I/R조관주4℃St.Thomas정도액정도,병재32℃하결혈40 min,재관주60 min;IPO조우재관주즉각행결혈후처리,재관주10 s,결혈10 s,공6개순배,연후회복관주58 min;M+IPO조우재관주즉각관주함N-(2-구기병선)-감안산2 mmol/L적K-H액3 min,연후행결혈후처리2min,재관주55 min.분별우평형관주말급재관주말기록HR、좌심실발전압(LVDP)、좌심실서장말압(LVEDP)화좌심실압력최대상승속도(+dp/dtmax).분별우재관주5 min화재관주말시,취좌심실심기조직,채용ELISA법측정ROS함량.우재관주말,취좌심실심기조직,관찰심기세포초미결구,병진행선립체손상평분,분별채용Western blot법화RT-PCR법검측심기조직Nrf2、혈홍소가양매-1(HO-1)、곤양화환원매1(NQO1)、초양화물기화매1(SOD1)급기mRNA적표체수평.결과 여C조비교,재관주말I/R조화M+IPO조HR、+dp/dtmax화LVDP강저,LVEDP승고,IPO조HR、LVDP강저,LVEDP승고(P＜0.05),HR화+dp/dtmax차이무통계학의의(P＞0.05),I/R조、IPO조화M+IPO조선립체손상평분승고,재관주말I/R조、IPO조화M+IPO조ROS함량승고,심기조직Nrf2、HO-1、NQO1화SOD1급기mRNA표체하조(P＜0.05).여I/R조비교,IPO조화M+IPO조재관주말HR、+dp/dtmax화LVDP승고,LVEDP화심기조직ROS함량강저,심기조직Nrf2、HO-1、NQO1화SOD1급기mRNA표체상조,IPO조선립체손상평분강저(P＜0.05),M+IPO조선립체손상평분차이무통계학의의(P＞0.05).여IPO조비교,M+IPO조재관주말HR、+dp/dtmax화LVDP강저,LVEDP화심기조직ROS함량승고,선립체손상평분승고,심기조직Nrf2、HO-1、NQO1화SOD1급기mRNA표체하조(P＜0.05).결론 결혈후처리가조절ROS적수평,격활Nrf2-ARE신호통로,감경대서심기결혈재관주손상.
Objective To investigate the relationship between the mechanism of ischemic postconditioning-induced activation of nuclear factor-E2 related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway during myocardial ischemia-reperfusion (I/R) and reactive oxygen species (ROS).Methods Healthy male Sprague-Dawley rats, aged 16-20 weeks, weighing 250-300 g, were heparinized and anesthetized with intraperitoneal 1％ pentobarbital sodium 40 mg/kg.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution.Thirty-two isolated rat hearts were randomly divided into 4 groups (n=8 each) using a random number table: control group (group C) , group I/R,ischemic postconditioning group (group IPO) , and N-(2-mercaptopropionyl)-glycine (a ROS scavenger) + IPO group (group M + IPO).After 20 min of equilibration, group C was continuously perfused with K-H solution for 100 min, and the isolated hearts received the drugs via the perfusion system in the other groups.Group I/R was perfused with cardioplegic solution 4 ℃ St.Thomas, and then was subjected to 40 min of ischemia at 32 ℃ followed by 60 min of reperfusion.In group IPO, ischemic postconditioning was induced by 6 cycles of 10 s reperfusion followed by 10 s limb ischemia starting from the onset of reperfusion, and the hearts were then perfused for 58 min.In group M + IPO, the hearts were perfused with K-H solution containing N-(2-mercaptopropionyl)-glycine 2 m mol/L for 3 min starting from the onset of reperfusion,underwent 2 min of ischemic postconditioning, and then was perfused for 55 min.Heart rate (HR), left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP),and positive maximal pressure of left ventricular increase (+dp/dtmax) were recorded at the end of equilibration and of reperfusion.At 5 min of reperfusion and the end of reperfusion, myocardial specimens were obtained from the left ventricle for determination of ROS content by enzyme-linked immunosorbent assay.At the end of reperfusion, myocardial specimens were obtained from the left ventricle for examination of the ultrastructure of myocardial cells and for determination of Nrf2, heme oxygenase-1 (HO-1) , quinone oxidoreductase 1 (NQO1), and superoxide dismutase 1 (SOD1) mRNA and protein expression (by using Western blot and real-time polymerase chain reaction).The damage to myocardial mitochondria was assessed using Flameng scoring.Results Compared with group C, HR, +dp/dtmax and LVDP were significantly decreased, and LVEDP was increased at the end of reperfusion in I/R and M+IPO groups, HR and LVDP were decreased, LVEDP was increased, and no significant changes were found in +dp/dtmax at the end of reperfusion in IPO group, Flameng score was increased in I/R, IPO and M+IPO groups , the ROS content was increased at the end of reperfusion in I/R, IPO and M+IPO groups, and Nrf2, HO-1,NQO1 and SOD1 mRNA and protein expression was down-regulated at the end of reperfusion in I/R, IPO and M+IPO groups.Compared with group I/R, HR, +dp/dtmax and LVDP were significantly increased, and LVEDP and ROS content were decreased at the end of reperfusion, Nrf2, HO-1, NQO1 and SOD1 mRNA and protein expression was up-regulated at the end of reperfusion in IPO and M+IPO groups, Flameng score was decreased in IPO group, there was no significant change in Flameng score in M+IPO group.Compared with group IPO, HR, +dp/dtmax and LVDP were significantly decreased, LVEDP and ROS content were increased at the end of reperfusion, Flameng score was increased, and Nrf2, HO-1, NQO1 and SOD1 mRNA and protein expression was down-regulated in M+IPO group.Conclusion Ischemic postconditioning can regulate ROS level and activate Nrf2-ARE signaling pathway, thus attenuating myocardial I/R injury in rats.
